EP0451810A1 - Conjugés de hapten-biotin et leur usage - Google Patents

Conjugés de hapten-biotin et leur usage Download PDF

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Publication number
EP0451810A1
EP0451810A1 EP91105678A EP91105678A EP0451810A1 EP 0451810 A1 EP0451810 A1 EP 0451810A1 EP 91105678 A EP91105678 A EP 91105678A EP 91105678 A EP91105678 A EP 91105678A EP 0451810 A1 EP0451810 A1 EP 0451810A1
Authority
EP
European Patent Office
Prior art keywords
biotin
hapten
mmol
theophylline
spacer
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
EP91105678A
Other languages
German (de)
English (en)
Other versions
EP0451810B1 (fr
Inventor
Erasmus Dr. Huber
Dietmar Dr. Zdunek
Christian Dr. Klein
Roland Dr. Schenk
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Roche Diagnostics GmbH
Original Assignee
Roche Diagnostics GmbH
Boehringer Mannheim GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Roche Diagnostics GmbH, Boehringer Mannheim GmbH filed Critical Roche Diagnostics GmbH
Publication of EP0451810A1 publication Critical patent/EP0451810A1/fr
Application granted granted Critical
Publication of EP0451810B1 publication Critical patent/EP0451810B1/fr
Anticipated expiration legal-status Critical
Expired - Lifetime legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F210/00—Copolymers of unsaturated aliphatic hydrocarbons having only one carbon-to-carbon double bond
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/555—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound pre-targeting systems involving an organic compound, other than a peptide, protein or antibody, for targeting specific cells
    • A61K47/557—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound pre-targeting systems involving an organic compound, other than a peptide, protein or antibody, for targeting specific cells the modifying agent being biotin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5306—Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)

Definitions

  • the invention relates to hapten-biotin conjugates and their use.
  • haptens which serve as parameters for certain diseases or the state of health of the human body. These include hormones, tumor markers and viral proteins. Most haptens are still assigned to most drugs, the determination of which is often necessary for the monitoring of drug treatment. Since all these haptens only occur in very small amounts, methods based on the principle of the immunoassay are used for their detection. There are many variations.
  • the various immunological determination methods can be divided into homogeneous and heterogeneous methods. In the heterogeneous processes, a solid phase reaction is always involved in order to separate the bound part of the labeled components from the unbound part. The labeling can be determined well in this type of process, but it is disadvantageous that the heterogeneous reaction takes a long time.
  • conjugated enzymes can be used as a label, which only acquire their enzyme activity if they are bound to the hapten or antigen to be determined or are activated by the substance to be determined.
  • conjugated enzymes can be used as a label, which only acquire their enzyme activity if they are bound to the hapten or antigen to be determined or are activated by the substance to be determined.
  • One more way consists in using a fluorescent substance as the label, the fluorescence of which is either shifted into another wave range or the polarization of which is changed by binding to the substance to be determined.
  • a homogeneous determination method was proposed in DE-A 38 22 750, in which the sample solution is incubated with 3 receptors R1, R2 and R3, of which R1 and R2 are capable of binding to one another and R3 is specifically bindable to the substance to be determined , wherein receptor R1 is a conjugate of a partner of a specific binding pair P and a substance S which corresponds to the substance to be determined or is a derivative thereof or has at least one epitope of the substance to be determined, R2 is a receptor which has at least two binding sites for the specific binding partner and R3 is a receptor which has at least two binding sites, at least one of which binds specifically to an epitope of the substance to be determined or of S.
  • receptor R1 is a conjugate of a partner of a specific binding pair P and a substance S which corresponds to the substance to be determined or is a derivative thereof or has at least one epitope of the substance to be determined
  • R2 is a receptor which has at least two binding sites for the
  • the substance to be detected competes with the receptor R1 for binding to receptor R3 and receptor R2 binds with receptor R1. Only when the receptors R1, R2 and R3 are bound does agglutination occur which can be monitored photometrically. The binding of the substance to be detected to receptor R3 prevents agglutination, so that the agglutination is an indirect measure of the content of the substance to be detected.
  • This method is suitable for the detection of immunologically active substances such as antigens, antibodies and haptens.
  • haptens a conjugate of a partner of a specific binding pair and a hapten is used as the receptor R1.
  • a conjugate of biotin and substance S is used as receptor R1, latex coated with streptavidin as receptor R2 and an antibody capable of binding to the substance to be detected is used as receptor R3.
  • the biotin-hapten conjugate binds to the latex coated with streptavidin via the biotin portion.
  • the antibody can bind with the hapten-biotin conjugate via the hapten portion. If two complexes of streptavidin-coated latex and biotin-hapten conjugate bind to the antibody, a turbidity occurs which can be evaluated. The greater the amount of analysis and the lower the solvation of the conjugate, the slower the turbidity occurs.
  • conjugates of a hapten and biotin must therefore be made available.
  • Hapten-biotin conjugates have long been known per se.
  • EP-A-315 317 describes a so-called bidentate conjugate which consists of an immunologically active molecule and a specifically binding partner which are connected via a spacer.
  • the conjugates are optimally effective when the spacer length is more than 22.2 ⁇ , which corresponds approximately to a chain length of 18 atoms, but that a chain length of more than 20 atoms in turn reduces the sensitivity.
  • the presence of more than 5 heteroatoms is disadvantageous.
  • no finally satisfactory results have yet been achieved with these conjugates.
  • hapten-biotin conjugates which are characterized in that the hapten is connected to the biotin via a spacer which has 26 to 40 atoms in the chain and contains at least 5 heteroatoms.
  • hapten-biotin conjugates are made available in which the hapten and the biotin molecule are connected via a spacer which has a chain length of 26 to 40 atoms and contains at least 5 heteroatoms.
  • the heteroatoms of the spacer can be the heteroatoms occurring in organic molecules, such as nitrogen, oxygen, sulfur, phosphorus, etc.
  • the spacer preferably contains nitrogen and oxygen atoms as heteroatoms.
  • the number the heteroatoms must be at least 5.
  • a higher proportion of heteroatoms is advantageous, the proportion of the heteroatoms being so large that every third atom in the spacer is a heteroatom.
  • a polyethylene oxide of the specified chain length can be used as a spacer.
  • the spacer length is in the range of 26 to 40 atoms, only the atoms in the chain being counted. Particularly advantageous results are obtained with spacers that have more than 30 atoms.
  • the conjugates according to the invention can be prepared either by reacting the hapten and the biotin with a bifunctional spacer molecule, the functional groups present on the hapten and in the biotin molecule reacting with the functional groups of the spacer molecule.
  • Another possibility is to derivatize the hapten and / or the biotin molecule and then, if appropriate, react the derivatives with a spacer molecule. The derivatives and spacer molecules are then again selected so that a spacer of the desired length and with the desired number of heteroatoms is formed.
  • Suitable as spacers are homofunctional or heterobifunctional linkers, such as dicarboxylic acids, diamines, amino acids, mercaptocarboxylic acids and halocarboxylic acids.
  • Spacers are preferably used which consist of succinate, glutarate, suberate, ethylenediamine, propylenediamine, 1,8-diamino-3,6-dioxaoctane, 1,12-diamino-4,9-dioxadodecane, aminobutyric acid, aminocaproic acid, thioglycolic acid, thiopropionic acid, bromoacetic acid and / or iodoacetic acid.
  • These synthesis building blocks must be assembled in such a way that a spacer of the desired length with the desired number of heteroatoms is formed.
  • Another object of the invention is the use of the hapten-biotin conjugates in a competitive homogeneous immunoassay, in which the agglutination occurring in the reaction is evaluated by turbidimetric or nephelometric measurements.
  • Biotin-DADOO-succ 238 mg (0.5 mmol) of Biotin-DADOO-succ is dissolved in 25 ml of absolute DMF and mixed with 57 mg (0.5 mmol) of N-hydroxysuccinimide and 69 ⁇ l (0.5 mmol) of 2- (4-morpholinyl) ethyl isocyanide (Merck, No. 818649). The mixture is stirred at 20 ° C. for 30 min, then 1.5 ml (10 mmol) of 1,8-diamino-3,6-dioxaoctane (DADOO, Messrs. Merck, No.
  • DADOO 1,8-diamino-3,6-dioxaoctane
  • Reagent 1 consisting of 0.1% by weight streptavidin latex (preparation according to EP-A 0 349 988)
  • Reagent 2 consisting of 1.5 x 10 ⁇ 6 mol / l monoclonal antibody against theophylline and 4.5 x 10 ⁇ 7 mol / l conjugate of theophylline and biotin (prepared according to Example 6) 3 ⁇ l sample are combined with 30 ml reagent 2 at 37 ° C and incubated for 5 min. Then 500 ⁇ l of reagent 1 streptavidin latex are added and the change in extinction is determined within 4.2 min. Table 1 shows the calibration values obtained using aqueous theophylline solutions as samples.
  • Reagent 1 consisting of 100 mmol / l barbiturate buffer pH 8.5, 2% by weight dextran sulfate, 0.02% by weight streptavidin latex (produced according to EP-A 0 349 988) 4 x 10 ⁇ 8 mol / l polyclonal antibodies (IgG against T4).
  • Reagent 2 consisting of 4 x 10 ⁇ 8 mol / l conjugate of T4 and biotin, produced analogously to Example 6, T anstelle- (tert-butyloxycarbonyl) -NHS (Boehringer Mannheim GmbH) T4 being used instead of theophillin-3cb-NHS .

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Food Science & Technology (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Epidemiology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Polymers & Plastics (AREA)
  • Heterocyclic Carbon Compounds Containing A Hetero Ring Having Oxygen Or Sulfur (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Nitrogen And Oxygen Or Sulfur-Condensed Heterocyclic Ring Systems (AREA)
  • Addition Polymer Or Copolymer, Post-Treatments, Or Chemical Modifications (AREA)
  • Peptides Or Proteins (AREA)
EP91105678A 1990-04-10 1991-04-10 Conjugés de hapten-biotin et leur usage Expired - Lifetime EP0451810B1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE4011601 1990-04-06
DE4011601A DE4011601A1 (de) 1990-04-10 1990-04-10 Hapten-biotin-konjugate und ihre verwendung

Publications (2)

Publication Number Publication Date
EP0451810A1 true EP0451810A1 (fr) 1991-10-16
EP0451810B1 EP0451810B1 (fr) 1995-08-16

Family

ID=6404148

Family Applications (1)

Application Number Title Priority Date Filing Date
EP91105678A Expired - Lifetime EP0451810B1 (fr) 1990-04-10 1991-04-10 Conjugés de hapten-biotin et leur usage

Country Status (8)

Country Link
US (1) US5219764A (fr)
EP (1) EP0451810B1 (fr)
JP (1) JPH082910B2 (fr)
KR (1) KR910018419A (fr)
AT (1) ATE126595T1 (fr)
DE (2) DE4011601A1 (fr)
DK (1) DK0451810T3 (fr)
ES (1) ES2077101T3 (fr)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994016330A1 (fr) * 1993-01-06 1994-07-21 Beckman Instruments, Inc. Procede de preparation d'un preconjugue
EP0599142A3 (fr) * 1992-11-24 1994-11-30 Merck Patent Gmbh Méthode de fabrication d'immunoconjugués.
WO1995032428A1 (fr) * 1994-05-23 1995-11-30 Beckman Instruments, Inc. Reactifs et procedes de dosage rapide et quantitatif d'agents pharmacologiques
WO1995033491A1 (fr) * 1994-06-06 1995-12-14 Elmaleh David R Composes de biotine pour cibler des tumeurs et des foyers d'infection
EP0640836A3 (fr) * 1993-08-24 1995-12-20 Wako Pure Chem Ind Ltd Méthode d'immunoessai.
WO2023083723A1 (fr) 2021-11-11 2023-05-19 F. Hoffmann-La Roche Ag Nouveaux anticorps monoclonaux dirigés contre l-thyroxine et leurs utilisations diagnostiques

Families Citing this family (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5534620A (en) * 1987-09-30 1996-07-09 Beckman Instruments, Inc. Method of heterogenous purification using a bidentate conjugate
US5536820A (en) * 1993-02-26 1996-07-16 E. I. Du Pont De Nemours And Company Avidin-binding azo reagents
US5527711A (en) * 1993-12-13 1996-06-18 Hewlett Packard Company Method and reagents for binding chemical analytes to a substrate surface, and related analytical devices and diagnostic techniques
US5716594A (en) * 1994-06-06 1998-02-10 The Jmde Trust Biotin compounds for targetting tumors and sites of infection
DE59607898D1 (de) † 1995-12-29 2001-11-15 Biotez Berlin Buch Gmbh Bioche Verfahren zur markierung von biomolekülen mit meerrettichperoxidase
DE19748489A1 (de) 1997-11-03 1999-05-06 Roche Diagnostics Gmbh Polyethylenglykol-derivatisierte Biomoleküle und deren Verwendung in heterogenen Nachweisverfahren
US6326479B1 (en) 1998-01-27 2001-12-04 Boston Probes, Inc. Synthetic polymers and methods, kits or compositions for modulating the solubility of same
US6153442A (en) * 1998-05-20 2000-11-28 Dade Behring Inc. Reagents and methods for specific binding assays
US20030003602A1 (en) * 2001-03-07 2003-01-02 Bernd Vogt Homogeneous immunoassay method
US7145019B2 (en) * 2003-10-16 2006-12-05 Ambergen, Inc. Photocleavable isotope-coded affinity tags
WO2007112079A2 (fr) * 2006-03-24 2007-10-04 Duke University Immunogène polyvalent
JP2010053118A (ja) * 2008-07-28 2010-03-11 Fujifilm Corp ビオチン化チロキシン
CN113721012B (zh) * 2021-08-27 2022-08-09 深圳上泰生物工程有限公司 一种组合物及其试剂盒在检测甘胆酸中的用途

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0183901A2 (fr) * 1984-11-27 1986-06-11 BEHRINGWERKE Aktiengesellschaft Haptènes bifonctionnels, procédé pour leur production et leur utilisation
EP0310361A2 (fr) * 1987-09-30 1989-04-05 Beckman Instruments, Inc. Conjugat tridenté et méthode de sa mise en oeuvre
EP0315317A2 (fr) * 1987-09-30 1989-05-10 Beckman Instruments, Inc. Conjugat bidenté et méthode de sa mise en oeuvre
EP0349988A2 (fr) * 1988-07-05 1990-01-10 Roche Diagnostics GmbH Méthode pour déterminer une substance spécifique capable de lier

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4617261A (en) * 1984-03-21 1986-10-14 Cetus Corporation Process for labeling nucleic acids and hybridization probes
NZ211453A (en) * 1984-03-22 1989-01-06 Biotechnology Research Enterpr Aryl azides, their preparation and use in the detection, localisation and isolation of polynucleotides
US4760142A (en) * 1984-11-27 1988-07-26 Hoechst Celanese Corporation Divalent hapten derivatives

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0183901A2 (fr) * 1984-11-27 1986-06-11 BEHRINGWERKE Aktiengesellschaft Haptènes bifonctionnels, procédé pour leur production et leur utilisation
EP0310361A2 (fr) * 1987-09-30 1989-04-05 Beckman Instruments, Inc. Conjugat tridenté et méthode de sa mise en oeuvre
EP0315317A2 (fr) * 1987-09-30 1989-05-10 Beckman Instruments, Inc. Conjugat bidenté et méthode de sa mise en oeuvre
EP0349988A2 (fr) * 1988-07-05 1990-01-10 Roche Diagnostics GmbH Méthode pour déterminer une substance spécifique capable de lier

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0599142A3 (fr) * 1992-11-24 1994-11-30 Merck Patent Gmbh Méthode de fabrication d'immunoconjugués.
WO1994016330A1 (fr) * 1993-01-06 1994-07-21 Beckman Instruments, Inc. Procede de preparation d'un preconjugue
EP0640836A3 (fr) * 1993-08-24 1995-12-20 Wako Pure Chem Ind Ltd Méthode d'immunoessai.
WO1995032428A1 (fr) * 1994-05-23 1995-11-30 Beckman Instruments, Inc. Reactifs et procedes de dosage rapide et quantitatif d'agents pharmacologiques
WO1995033491A1 (fr) * 1994-06-06 1995-12-14 Elmaleh David R Composes de biotine pour cibler des tumeurs et des foyers d'infection
WO2023083723A1 (fr) 2021-11-11 2023-05-19 F. Hoffmann-La Roche Ag Nouveaux anticorps monoclonaux dirigés contre l-thyroxine et leurs utilisations diagnostiques

Also Published As

Publication number Publication date
EP0451810B1 (fr) 1995-08-16
DE4011601A1 (de) 1991-10-17
ES2077101T3 (es) 1995-11-16
JPH04224583A (ja) 1992-08-13
DK0451810T3 (da) 1996-01-02
JPH082910B2 (ja) 1996-01-17
US5219764A (en) 1993-06-15
DE59106246D1 (de) 1995-09-21
KR910018419A (ko) 1991-11-30
ATE126595T1 (de) 1995-09-15

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